JPH05284994A - Anti-human pivka-ii antibody, hybridoma and method for immunological measurement - Google Patents

Abstract

PURPOSE:To obtain a new monoclonal antibody useful for immunological determination of human PIVKA-II. CONSTITUTION:A monoclonal antibody (immunoglobulin class is IgG) specifically reacting with human PIVKA-II but not human prothrombin and human thrombin. The monoclonal antibody is obtained by culturing a hybridoma of mammalian spleen cells immunized against artificial human PIVKA-II prepared by decarboxylating human prothrombin and mammalian myeloma cells.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to human PIVKA-II.
(Protein induced by vitamin K absense-II) -reactive various monoclonal antibody groups, hybridoma groups secreting these monoclonal antibodies, and human PIVK using these monoclonal antibody groups
The present invention relates to an immunological quantification method of A-II.

[0002]

2. Description of the Related Art Prothrombin is a glycoprotein also called blood coagulation factor II.
It has γ-carboxyglutamic acid residues (hereinafter referred to as Gla). When liver parenchymal cell damage due to hepatocellular carcinoma, vitamin K deficiency, vitamin K antagonist administration, etc., some or most of the 10 Glas of this prothrombin are glutamic acid residues with incomplete carboxylation. It is released into the blood as it is (hereinafter referred to as Glu).
This glycoprotein is identified as abnormal prothrombin or PIV
It is called KA-II. In recent years, it has been reported that PIVKA-II is expressed at a high rate in hepatocellular carcinoma patients and appears at a high concentration in plasma as compared with healthy subjects, and it is a new tumor marker for hepatocellular carcinoma or a monitor for diagnosis. It is regarded as important as a marker for. Therefore, it is necessary to accurately and simply measure the amount of PIVKA-II in blood or plasma.

The following four types of methods can be mentioned as typical methods of measuring PIVKA-II known in the prior art. The first method is a method using a two-dimensional cross immunoelectrophoresis method. The second method is PIVKA-II.
This is a competitive radioimmunoassay using a polyclonal antibody specific for. The third method is to adsorb and remove prothrombin mixed with barium sulfate, and measure PIVKA-II by an indirect agglutination method using latex. The fourth method is
This is an enzyme-linked immunosorbent assay in which both a monoclonal antibody that specifically recognizes PIVKA-II and a polyclonal antibody against prothrombin are used, and one of them is used as an immobilized antibody and measured as an enzyme-labeled antibody.

However, the first method has the drawback of low sensitivity and lack of quantitativeness. The second method is a facility problem of using radioisotopes, and PIV.
Purification of a polyclonal antibody specific for KA-II is complicated, and there are various problems that there are lot differences. Third
In the above method, since the antibody used in the indirect agglutination method is a polyclonal antibody against prothrombin, PIVKA-
There is a drawback that II cannot be measured specifically. In the fourth method, since the monoclonal antibody is used, the drawbacks of the first and second methods are improved in that the antibody is excellent in specificity and the antibody can be stably supplied. However, it does not solve the problem that it is complicated and takes a long time for measurement.

[0005]

DISCLOSURE OF THE INVENTION The present inventor has found that PIVK
As a result of intensive research to develop a method for easily and accurately and reproducibly measuring A-II, the first group of monoclonal antibodies that show specific reactivity to human PIVKA-II but do not react to human prothrombin. (GLA-1, GLA
-2), a second monoclonal antibody group (T-, which shows reactivity to both human thrombin and human prothrombin).
1, T-2), a third monoclonal antibody group (PT-1, PT-2) that reacts with human prothrombin but does not react with human thrombin was found. First of the group
Of the PIVKA-II in the plasma rapidly and accurately by using the combination of the two types of the monoclonal antibody group and the second or third monoclonal antibody group and the three types of the first, second and third monoclonal antibody groups. , B / F separation was not required, and it was found that specific measurement can be performed without being interfered with by prothrombin, which is present in a large amount in the sample as compared with PIVKA-II. Therefore, an object of the present invention is to provide the above novel monoclonal antibody, hybridoma cells producing the monoclonal antibody, and an immunological quantification method using the monoclonal antibody.

[0006]

SUMMARY OF THE INVENTION Therefore, the present invention provides
(1) A monoclonal antibody that specifically reacts with human PIVKA-II and does not react with human prothrombin and human thrombin, (2) a human PIVKA-II, a monoclonal antibody that reacts with human thrombin and human prothrombin, and (3) It relates to a monoclonal antibody that specifically reacts with human PIVKA-II and human prothrombin, but does not react with human thrombin. Further, the present invention is formed by fusing a spleen of a mammal immunized with an artificial human PIVKA-II prepared by decarboxylation of human prothrombin and a myeloma cell of the mammal, wherein each of the monoclonal antibodies (1), It also relates to each hybridoma secreting (2) or (3) or a cell line derived therefrom. Furthermore, in the present invention, at least one of the monoclonal antibody (2) or the monoclonal antibody (3) and the monoclonal antibody (1) immobilized on an insoluble carrier are brought into contact with a test sample to obtain a test sample. Human PIVK characterized by observing agglutination in
It also relates to a method for measuring A-II.

The present invention will be described below in the order of a monoclonal antibody, a hybridoma and an immunological measuring method. Artificial PIVKA-II used as an immunogen is described in, for example, P.
A. Price method (J. Biol. Chem. 25.
4,431,1979). Then, the purified artificial PIVKA-II immunogen solution is used to immunize a mammal (eg, mouse, rat, rabbit, goat or horse) by in vivo immunization.

[0008] Specifically, for example, purified artificial PIV
The KA-II immunogen solution is mixed with an equal volume of Freund's complete or incomplete adjuvant until emulsified. This mixed solution is subcutaneously administered to, for example, a mouse (first immunization). Thereafter, the same operation is performed at intervals of 2 to 4 weeks, and immunization is performed several times. A few days after the final immunization, the spleen is aseptically removed, crushed with a stainless mesh or the like to prepare spleen cells, and used in the cell fusion step.

As the other parent cell used for cell fusion, myeloma cells (myeloma cells), various known cell lines such as p3 (p3 / x63-Ag8) [Na
, 256, 495-497 (1975)], p.
3-U1 [CurrentTopics in Mic
robbiology and Immunology,
81; 1-7 (1978)], NS-1 [Eur. J.
Immunol. , 6; 511-519 (197).
6)], MPC-11 [Cell, 8; 405-415]
(1976)], SP2 / 0 [Nature, 276;
269-270 (1978)], FO [J. Immun
ol. Meth. , 35; 1-21 (1980)], ×
63.6.55.3 [J. Immunol. , 123;
1548-1550 (1979)], S194 [J. E
xp. Med. , 148; 313-323 (197).
8)], or R210 [Natur in rats]
e, 277; 131-133 (1979)] and the like.

The cell fusion between the immune spleen cells and the myeloma cells can be carried out by a conventional method. For example, a known fusion promoter (polyethylene glycol, Sendai virus, etc.) is used, and in some cases an auxiliary agent (dimethyl sulfoxide, etc.) is used. Can also be used. The use ratio of immune spleen cells and myeloma cells may be the same as in the conventional method, for example,
Spleen cells are used in an amount of about 1 to 10 times that of myeloma cells. The fusion medium is, for example, 40% (w
/ V) Dulbecco's modified Eagle medium (DMEM) containing polyethylene glycol can be used. The fusion is performed by thoroughly mixing the immune spleen cells and myeloma cells in the above medium. Subsequently, cells other than the hybridomas are removed using a selection medium (for example, HAT medium), and the presence or absence of antibody production in the hybridoma culture supernatant is measured by, for example, the ELISA method to isolate the target hybridoma.

The thus obtained hybridomas secreting each of the monoclonal antibodies of the present invention (described later) can be subcultured in an ordinary medium and can be easily stored in liquid nitrogen for a long period of time. it can.

As the medium for culturing the hybridoma, any medium suitable for culturing the hybridoma can be used, and preferably DMEM has fetal bovine serum, L-glutamine, L-pyruvate and antibiotics. A medium containing (penicillin G and streptomycin) is used.

In the case of in vitro culture, the above hybridomas are cultured, for example, in a medium at a concentration of 5% CO _{2 and at a} concentration of 3%.
It is preferably carried out at 7 ° C. for about 3 days, and for about 14 days when cultured in vivo, for example, in the abdominal cavity of a mouse.

The desired monoclonal antibody is isolated from the culture medium in which the above hybridoma is cultured by a conventional method, or from the ascites of a suitable mammal (eg, mouse or rat) to which any of the above six hybridomas has been administered. , Can be purified.

When the monoclonal antibody is separated and purified from the thus-produced culture medium or mouse ascites fluid, it is possible to use a method generally used for protein isolation and purification. Examples of such a method include salting out with ammonium sulfate, ion exchange chromatography, molecular sieve column chromatography using a molecular sieve gel, affinity column chromatography using a protein A-bound polysaccharide, dialysis, and freeze drying.

The thus obtained monoclonal antibody of the present invention can be classified into the following three types depending on its reactivity. (1) A first group of monoclonal antibodies that specifically react with human PIVKA-II and do not react with human prothrombin (for example, monoclonal antibodies GLA-1 and GLA).
-2). (2) A second monoclonal antibody group that specifically reacts with human thrombin and human prothrombin (for example, monoclonal antibodies T-1 and T-2). (3) A third group of monoclonal antibodies (for example, monoclonal antibodies PT-1 and PT-2) that specifically react with human prothrombin and do not react with human thrombin.

The first to third monoclonal antibody groups according to the present invention are immobilized on an insoluble carrier, and the first monoclonal antibody and the second and / or third monoclonal antibodies are contacted with a test sample. Then, agglutination reaction does not occur with prothrombin in the test sample, and P
Since it can cause an agglutination reaction only with IVKA-II, it can be used in the immunological quantification method of PIVKA-II, and is also useful as a reagent for immunological quantification.

The test sample used in the immunological quantification method of the present invention is not particularly limited as long as it is a sample that may contain PIVKA-II. For example, a biological sample, particularly blood, Plasma or urine, preferably plasma. In the immunological quantification method of the present invention, even if the test sample is used as it is without being diluted, the interference of human prothrombin existing in a free state in the test sample can be avoided.

As the insoluble carrier, any insoluble carrier generally used in an immunological measuring method utilizing the agglutination reaction of antigen-antibody can be used. For example, latex particles (particularly polystyrene latex particles) can be used. Can be mentioned.

To immobilize the monoclonal antibody according to the present invention on an insoluble carrier, a known method such as a chemical binding method (using carbodiimide, glutaraldehyde or the like as a crosslinking agent) or a physical adsorption method can be used. Thus, a complex of the monoclonal antibody and the insoluble carrier is formed, which can be used in the immunological quantification method of the present invention.

In the immunological quantification method of the present invention, at least two kinds of monoclonal antibodies immobilized on the insoluble carrier are used (the first monoclonal antibody is essential). Two or three kinds of complexes prepared by immobilizing a monoclonal antibody on an insoluble carrier are used, or a complex prepared by immobilizing two or three kinds of monoclonal antibodies on a certain insoluble carrier is used. be able to. Further, a combination of one complex prepared by immobilizing a certain one kind of monoclonal antibody on an insoluble carrier and one complex prepared by immobilizing two kinds of monoclonal antibodies on a certain one insoluble carrier. Can also be used. As a combination of two kinds of monoclonal antibodies of the present invention, a combination of the above-mentioned first monoclonal antibody (GLA-1, GLA-2) and second monoclonal antibody (T-1, T-2) is preferable.

In the assay method of the present invention, a known fixed amount of the above-mentioned monoclonal antibody-immobilized insoluble carrier complex and a fixed amount of an aqueous test sample containing an unknown amount of PIVKA-II are used in a suitable reaction container (eg, , On a slide plate or in a reaction cell). For example, in the case of a plasma sample, 1 part by volume of the plasma sample (non-diluted solution) is added with 1 to 3 parts by volume of the complex suspension water (concentration of 1% or more) and brought into contact. Further, in order to make the agglutination image clearer, a buffer solution (for example, Tris-hydrochloric acid buffer solution) may be added to the sample and the complex-suspended water to bring them into contact with each other. The PIVKA-II concentration can be quantified based on the degree of aggregation thus formed. This agglutination reaction is not disturbed by the free prothrombin present in plasma samples. For example, the agglutination reaction is measured visually on a slide plate and spectroscopically using a specific wavelength in the case of a reaction cell to determine the PIV in the test sample.
The KA-II concentration can be quantified.

[0023]

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples. Example 1: Preparation of artificial PIVKA-II (a) Preparation of artificial PIVKA-II was carried out from purified prothrombin by P. A. Price method (J. Biol. C
hem. 254, 431, 1979).
That is, 3 mg of human prothrombin was dissolved in 0.05M hydrochloric acid and 0.1M NH ₄ HCO ₃ and freeze-dried.
The dried prothrombin was heated at 110 ° C. under reduced pressure and then purified by gel chromatography on Sephadex G-100 (4 ° C.).

(B) Preparation of immunized spleen cells Artificial PIVKA-II immunogen solution (A280nm = 0.
1) was mixed with an equal amount of Freund's complete adjuvant until emulsified, and 200 μl of the mixed solution was intraperitoneally administered to BALB / c mice to immunize (first).
Immunity). After 30 days, 200 μ of the same mixed solution as above
1 was intraperitoneally administered to the mouse (second immunization).
21 days after the second immunization, 200 μl of the artificial PIVKA-II diluent prepared by diluting the artificial PIVKA-II immunogen solution (A280 nm = 0.1) with an equal amount of physiological saline.
Was administered intravenously to the mice (final immunization). Three days after the final immunization, the spleen was aseptically removed from the mouse and used in the next cell fusion step.

(C) Cell fusion The aseptically extracted spleen was placed in a petri dish containing 5 ml of DME medium containing 15% fetal bovine serum. Next, the spleen was refluxed with about 15 ml of DME medium containing 15% fetal bovine serum to allow spleen cells to flow out, and the spleen cell suspension was passed through a nylon mesh. The spleen cells were collected in a 50 ml centrifuge tube and centrifuged at 500 xg for 10 minutes. The pellet thus obtained was hemolyzed (155 mM-NH ₄ Cl, 10 mM-KHCO ₃ , 1
4 ml of mM-Na ₂ EDTA: pH 7.0) was added and suspended. It was left at 0 ° C. for 5 minutes to destroy the red blood cells in the suspension. DME medium containing 10 ml of 15% fetal bovine serum was added, followed by centrifugation. The pellet thus obtained was washed with DME medium by centrifugation, and the number of living spleen cells was measured.

On the other hand, 1 × 10 ⁸ spleen cells were added to approximately 2 × 10 ⁷ cells of mouse myeloma cells (myeloma cells) SP2 / 0-Ag14 (RIKEN Gene Bank Cell Bank) that had been cultured in advance. , Mix well in DME medium,
Centrifugation was performed (500 xg, 10 minutes). The supernatant is aspirated, the pellet is thoroughly disentangled, and 40% polyethylene glycol 4000 solution (incubated at 38 ° C) 0.5 m
l was added dropwise and the centrifuge tube was gently rotated by hand for 1 minute to mix the polyethylene glycol solution with the cell pellet. Next, D kept warm at 38 ° C
1 ml of ME medium was added every 30 seconds, and the tube was gently rotated. After repeating this operation 10 times, 1
Add DME medium containing 20 ml of 5% fetal bovine serum,
Centrifugation (500 xg, 10 minutes) was performed. After removing the supernatant, HAT medium containing 15% fetal bovine serum (DM
Aminopterin 4 × 10 ⁻⁷ M, thymidine 1.6 in E medium
The cell pellet was washed twice by centrifugation with 10 × 10 ⁻⁵ M and 1 × 10 ⁻⁴ M of hypoxanthine) and then suspended in 40 ml of the HAT medium. Add 2 to each well of the 96-well cell culture plate.
Aliquots of 00 μl were dispensed, and the culture was started at 37 ° C. in a carbon dioxide incubator containing 5% carbon dioxide. During the culture, about 100 μl of the medium in each well was removed at intervals of 2 to 3 days, and the H
Hybridomas growing in HAT medium were selected by adding 100 μl of AT medium. 15% from the 8th day
Change to HAT medium containing fetal bovine serum (DME medium supplemented with thymidine of 1.6 × 10 ⁻⁵ M and hypoxanthine of 1 × 10 ⁻⁴ M), and observe the hybridomas. Then, the anti-artificial PIVKA-II antibody-producing hybridomas were screened by the ELISA method described below.

(D) Establishment of hybridoma The presence or absence of the produced antibody in the supernatant of the hybridoma culture solution was measured by the ELISA method. The artificial PIVKA-II immunogen solution (A280 nm = 0.05, diluted with physiological saline) was added to each well of a 96-well ELISA plate (Immulon 1.1., Nippon Dynatech Co., Ltd.).
50 μl of each was dispensed and left at 25 ° C. for 2 hours. Next, after washing three times with 0.05% Tween20-physiological saline, 50 μl of the culture supernatant was added to each well and the temperature was adjusted to 25 ° C.
And reacted for 1 hour.

Next, 20 with Tween 20-saline.
50 μl of 0-fold diluted peroxidase-conjugated anti-mouse antibody (Dako, Denmark) was added to each well. After completion of the reaction, each well was washed 3 times with 0.05% Tween20-saline, and 0.5 mM aminoantipyrine, 1 mM was added.
250 μl of a solution containing 0 mM phenol and 0.005% hydrogen peroxide was added to each well and reacted at 25 ° C. for 30 minutes, and the absorbance at 490 nm of each well was measured. As a result, antibody production was observed in 6 of the 192 wells. 24 hybridomas in 6 wells
Transfer to a well plate and HA containing 15% fetal bovine serum
The cells were cultured in T medium for 4 to 5 days. After that, ELISA again
The presence or absence of production of anti-artificial PIVKA-II antibody was confirmed by the method before cloning by the limiting dilution method. The limiting dilution method is 96 wells in which 2 × 10 ⁴ peritoneal cells of normal BALB / c mouse per well are preliminarily injected with a cell suspension diluted with HT medium to 5 hybridomas / ml. 100 μl was dispensed to each well of the plate. 10 days later, artificial PIVK by ELISA method
Hybridoma clones producing A-II specific antibodies were screened.

As a result, 20 for each hybridoma
-40 antibody producing clones were obtained. From these clones, a clone having a strong proliferative ability, a high antibody secretory ability, and a stable one was selected, and recloned in the same manner as described above to obtain 6 kinds of anti-artificial PIVKA-II antibody-producing hybridoma GLA-. 1, GLA-2, T-1, T-2,
And PT-1 and PT-2 were established. Six monoclonal antibodies GLA-1, GLA-2, T-1, T-2, and PT secreted from these six hybridomas
The reactivity of -1, PT-2 with human prothrombin or human thrombin (Atens Research Co., USA) was determined by coating a 96-well ELISA plate with human thrombin or human prothrombin,
It investigated by the method similar to the method A. Monoclonal antibody G
LA-1 and GLA-2 did not react with both human thrombin and human prothrombin. Monoclonal antibodies T-1 and T-2 reacted with both human thrombin and human prothrombin. Meanwhile, monoclonal antibody PT-1,
PT-2 responded to human prothrombin but not human thrombin.

Example 2: Production of monoclonal antibody (a) In vitro method Mouse hybridomas GLA-1, GLA-2, T-
1, T-2, and PT-1 and PT-2 are 1
The cells were cultured in a DME medium containing 5% fetal bovine serum at 37 ° C. in a 5% carbon dioxide atmosphere for 72 to 96 hours. After centrifuging the culture (10000 × g for 10 minutes), solid ammonium sulfate was gradually added to the supernatant to a final concentration of 50%. The mixture was stirred under ice cooling for 30 minutes, then left for 60 minutes and then centrifuged (10000 xg,
10 minutes), the precipitate obtained is dissolved in a small amount of 10 mM phosphate buffer (pH 8.0), and 1000 times the volume of 10
A column of DEAE-cellulose was previously equilibrated with mM phosphate buffer. Elution of the monoclonal antibody was performed with 10 mM phosphate buffer (pH 8.0) and 0.2 M-Na.
The concentration gradient method was performed between 10 mM phosphate buffer (pH 8.0) containing Cl. The eluted monoclonal antibody was concentrated by the ultrafiltration method, and 0.1M phosphate buffer (pH
It was dialyzed against 8.0). To remove the bovine serum IgG, dialyzate the goat anti-bovine serum IgG-Sepharose 4B.
Passed through the column. Next, the flow-through was loaded onto a Protein A-Sepharose 4B column equilibrated with 0.1 M phosphate buffer (pH 8.0). The column was eluted with a buffer solution having a pH of 3.5, and purified anti-human PIVKA-II specific monoclonal antibody GLA-1, and similarly monoclonal antibody G
A solution of LA-2, monoclonal antibody T-1, monoclonal antibody T-2, monoclonal antibody PT-1 and monoclonal antibody PT-2 was obtained.

(B) In Vivo Method 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane) was intraperitoneally administered to 10-12 week old BALB / c mice, and then 14-20. Hybridomas GLA-1, GLA-2, T-1, T-2, PT-1, proliferated in vitro in mice on day 1
Alternatively, PT-2 was inoculated at 2 × 10 ⁶ per mouse.

About 10 to 15 ml of ascites was obtained from one mouse for each hybridoma. The antibody concentration is
It was 2-10 mg / ml. Purification of the monoclonal antibody in the ascites was performed by the same method as the above-mentioned in vitro purification (except the operation of passing a goat anti-bovine serum IgG-Sepharose 4B column).

Example 3: Identification of immunoglobulin class and specificity of monoclonal antibodies Monoclonal antibodies GLA-1, GLA-2, T-1,
Identification of the immunoglobulin class and specificity of T-2, PT-1, or PT-2 was carried out by the Octelony immunodiffusion method and the enzyme immunoassay method, respectively.
The results are shown in Tables 1 and 2.

[0034]

[Table 1]

[0035]

[Table 2]

In Tables 1 and 2, + means a reaction by the ELISA method, and-means a reaction by the ELISA method. In addition, as shown in FIG. 1, in the amino acid sequences 1 to 582 of prethrombin, prethrombin 1 is 158 to 582, fragment 1 is 1 to 157, and fragment 2 is 158 to 2
75th, 1st to 41st glazed area,
The 24th position (however, a synthetic peptide in which gla is replaced by glu) and the peptide II are 25th to 41st positions (however, gla)
Is a synthetic peptide).

Example 4: Binding of antibody to insoluble carrier (latex) 2 ml of an aqueous solution containing the monoclonal antibody GLA-1 (2.0 mg / ml) and a latex solution (2%, Do)
w Chemical: particle size 0.482 μm) 2 ml
And were mixed and stirred for about 1 hour. Centrifuge (20,000 x
(g, 10 minutes), the precipitate was suspended in a 0.1% BSA solution and stirred for about 1 hour. Centrifuge again (20,000 xg,
After 10 minutes), the precipitate was suspended in water and stirred for about 2 hours. Thus, a liquid containing the monoclonal antibody GLA-1 / latex complex was obtained. Similarly, using the monoclonal antibody GLA-2, the monoclonal antibody T-1, the monoclonal antibody T-2, the monoclonal antibody PT-1 and the monoclonal antibody PT-2, a solution containing a complex of each individual monoclonal antibody and latex. Was prepared.

A complex of the antibody mixture and latex was prepared as follows. Monoclonal antibody GLA-1, monoclonal antibody T-1 and monoclonal antibody PT
2 ml of an aqueous solution containing 0.66 mg / ml each of -1 and a latex solution (2%, Dow Chemi
cal company: particle size 0.482 μm) 2 ml was mixed and stirred for about 1 hour. Thereafter, the same treatment as described above is carried out to carry out monoclonal antibody GLA-1 / monoclonal antibody T-1 /
A monoclonal antibody PT-1 / latex complex was prepared. Other combinations were prepared in a similar manner.

Monoclonal antibody GLA-1 / was prepared in the same manner as described above except that an aqueous solution containing 1 mg / ml each of monoclonal antibody GLA-1 and monoclonal antibody T-1 and an equal amount of the latex solution were mixed.
A monoclonal antibody T-1 / latex complex was prepared.

Example 5: Quantification by slide agglutination reaction 30 μl of antibody latex complex-containing solution prepared in Example 4
1 and an aqueous solution containing PIVKA-II in various concentrations 30
μl was mixed on a slide glass, and after shaking for 3 minutes, an aggregated image was visually judged. The results are shown in Tables 3 to 6 below.

In Tables 3 to 6, + means cohesion, and-means no coagulation. Also, the antibodies in each table /
In the latex complex column, the type of complex is indicated by the monoclonal antibody that binds to the complex. Thus, for example, GLA-1 is the monoclonal antibody GLA-1.
/ Latex complex, and GLA-1 + T-1 means a mixture of equal amounts of the monoclonal antibody GLA-1 / latex complex and the monoclonal antibody T-1 / latex complex. Furthermore, GLA-1 / T-1 is a monoclonal antibody GLA-1 containing liquid, monoclonal antibody T-1.
It is a mixture of the containing liquid and the latex solution in equal amounts.

[0042]

[Table 3]

[0043]

[Table 4]

[0044]

[Table 5]

[0045]

[Table 6]

Example 6 Addition and Recovery Test of Purified PIVKA-II Five kinds of samples, namely, plasma collected from healthy person A (sample 1), plasma collected from healthy person B (sample 2), hepatocellular carcinoma patient C The PIVKA-II concentration in plasma (Sample 3) collected from hepatocellular carcinoma patient D, plasma (Sample 4) collected from hepatocellular carcinoma patient D, and plasma (Sample 5) collected from hepatocellular carcinoma patient E was determined using the monoclonal antibody of Example 5. It was measured using GLA-1 / monoclonal antibody T-1 / latex complex solution.
Next, purified PIVKA-II was added to each sample in an amount described in Table 7, and an addition recovery test was conducted. The measurement value was semi-quantitatively measured from the dilution ratio at which the specimen was diluted twice and the aggregation disappeared. The results are shown in Table 7, and good recovery was obtained.

[0047]

[Table 7]

Example 7: PI of healthy subjects and patients with hepatocellular carcinoma
VKA-II value Using the monoclonal antibody GLA-1 / monoclonal antibody T-1 / latex complex solution used in Example 6, the PIVKA-II amount of 20 healthy subject plasma and 55 hepatocellular carcinoma patient plasma samples was measured. did. Results are shown in FIG. The amount of PIVKA-II in the healthy group was less than 0.1 μg / ml in all cases. In contrast, the hepatocellular carcinoma patient group was 1 μg /
It was more than ml.

[0049]

INDUSTRIAL APPLICABILITY According to the present invention, the amount of PIVKA-II in patient plasma can be specifically determined without being interfered with by prothrombin in plasma, even if the plasma sample is not diluted.
It can be measured simply and quickly by the agglutination method.
This was made possible for the first time by the present invention. Therefore, the present invention provides a useful means for the diagnosis and pathological study of hepatocellular carcinoma and the like.

[Brief description of drawings]

FIG. 1 is an explanatory view showing the structures of various peptide fragments used in identifying the reaction specificity of each monoclonal antibody according to the present invention.

FIG. 2 is an explanatory diagram showing the results of measuring the amount of PIVKA-II in plasma of healthy subjects (20 samples) and plasma of patients with hepatocellular carcinoma (55 samples) using the monoclonal antibody latex complex of the present invention.

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Claims (7)

[Claims] 1. A specific reaction with human PIVKA-II,
A monoclonal antibody that does not react with human prothrombin and human thrombin. 2. A monoclonal antibody according to claim 1, which is formed by fusion of spleen of a mammal immunized with artificial human PIVKA-II prepared by decarboxylation of human prothrombin and mammalian myeloma cells, and secreting the monoclonal antibody. A hybridoma or a cell line derived from it. 3. A monoclonal antibody reactive with human PIVKA-II, human thrombin and human prothrombin. 4. A monoclonal antibody according to claim 3, which is formed by fusion of spleen of a mammal immunized with artificial human PIVKA-II prepared by decarboxylation of human prothrombin and mammalian myeloma cells, and secretes the monoclonal antibody of claim 3. A hybridoma or a cell line derived from it. 5. A monoclonal antibody that specifically reacts with human PIVKA-II and human prothrombin, but does not react with human thrombin. 6. The monoclonal antibody according to claim 5, which is formed by fusion of spleen of a mammal immunized with artificial human PIVKA-II prepared by decarboxylation of human prothrombin and mammalian myeloma cells, and secreting the monoclonal antibody according to claim 5. A hybridoma or a cell line derived from it. 7. A test sample is contacted with at least one of the monoclonal antibody according to claim 3 or the monoclonal antibody according to claim 5 and a monoclonal antibody according to claim 5, which are immobilized on an insoluble carrier, Human PI characterized by observing agglutination reaction in a test sample
VKA-II measurement method.